The 119 kDa and 124 kDa polyproteins of arabis mosaic nepovirus (isolate S) are encoded by two distinct RNA2 species.

Arabis mosaic virus (ArMV) is a nepovirus that is serologically distantly related to grapevine fanleaf virus (GFLV). Both ArMV and GFLV induce grapevine degeneration disease. Several ArMV isolates, unlike isolates of GFLV, produce upon in vitro translation of RNA2 a polyprotein (P2) that forms a double band in polyacrylamide-SDS gels. Cloning of full-length copies of RNA2 of an ArMV isolate from grapevine (ArMV-S) revealed that this isolate contained two RNA2s of different length, called RNA2-U and RNA2-L. The two species were not readily separated by electrophoresis of the virion RNA under denaturing gel electrophoresis conditions but could be distinguished by analysis of primer extension and in vitro translation products. The size difference of the two RNA2s is due mostly if not exclusively to differences in their coding regions. The 124 kDa RNA2-U-encoded polyprotein P2' and the 119 kDa RNA2-L-encoded polyprotein P2", which co-migrate, respectively, with the upper and lower polyprotein bands produced by RNA2 of ArMV-S, were more than 95% identical except in their N-terminal domains. In vitro maturation experiments and sequence comparisons indicate that the N-terminal products of P2' and P2" have a molecular mass of 31 kDa and 26 kDa. The genomic organization proposed is similar to that of GFLV RNA2.

Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo.

The putative 38 kDa movement protein (P38) gene, located on the RNA2 of grapevine fanleaf nepovirus (GFLV), was cloned in Escherichia coli and expressed as a fusion protein fused to six histidines (6HisP38). This protein was purified and used to produce a specific polyclonal antiserum from which immunoglobulins were isolated by immunoaffinity against the recombinant 6HisP38. Western immunoblot analyses on GFLV RNA2 in vitro maturation products showed that the antibodies were specific for P38 protein. This protein was detected as early as 18 h post-inoculation in GFLV-infected Chenopodium quinoa protoplasts and accumulated to very high levels. Tissue-prints and time course experiments on infected C. quinoa plants confirmed that P38 is present at a high level late in infection and is a final maturation product of the GFLV RNA2 polyprotein in vivo. P94 and P66 intermediates of maturation and polyprotein P2 were also detected in vivo but in very low concentrations. No significant difference was observed in the relative amounts of P66 and P94 detected in vivo, contrary to what occurs in vitro. Subcellular fractionation studies showed that P38, although mainly cytosolic, is also found in association with cell wall and membranes. Thought to be the GFLV movement protein, P38 would thus behave in an 'atypical' manner.

Differential proteolytic activities of precursor and mature forms of the 24K proteinase of grapevine fanleaf nepovirus.

The presence of a genome-linked protein (VPg) at the RNA 5'-end of the genome is a characteristic of different groups of animal and plant positive-sense single-stranded RNA viruses. These viruses express their structural and functional proteins from polyproteins that are sequentially processed by at least one viral proteinase. The grapevine fanleaf nepovirus 24K chymotrypsin-like cysteine proteinase, located between the VPg and the RNA polymerase in the RNA-1 encoded polyprotein P1, is active in its free form and in various precursors forms. The VPg proteinase precursor (VPg-Pro) constitutes a stable protein and its maturation in the reticulocyte lysate system occurs at a very low rate. Differences on cleavage activity were observed between the proteinase and its VPg-Pro precursor forms, depending upon the cleavage site considered. The proteinase alone has a greater cleavage efficiency than VPg-Pro at the Arg605/Gly606 and Cys257/Ala258 sites of polyprotein P2. On the other hand, the presumed Cys415/Ala416 site, present at the amino terminus of polyprotein P1, was preferentially cleaved by the VPg-Pro precursor. During their in vitro maturation, proteins containing the VPg proteinase-polymerase coding region or the proteinase-polymerase region were similar in their ability to cleave in cis between the proteinase and the RNA polymerase.

Genome organization of grapevine fanleaf nepovirus RNA2 deduced from the 122K polyprotein P2 in vitro cleavage products.

The full-length transcript of grapevine fanleaf virus (GFLV) RNA2 produces a primary product of 122K when translated in the rabbit reticulocyte system. This 122K polyprotein is completely processed in vitro by the RNA1-encoded 24K proteinase. The positions of the cleavage sites within the polyprotein have been mapped and the genome organization of GFLV-F13 RNA2 has been established. The order of mature proteins in the 122K polyprotein is the amino-terminal 28K protein, the 38K protein followed by the 56K coat protein at the carboxy terminus. These proteins represent the final cleavage products of the 122K polyprotein. A 66K protein which yields 28K and 38K proteins constitutes the major maturation intermediate. Microsequencing of the amino extremity of radioactively labelled 38K protein allowed identification of the Cys257/Ala258 site as the cleavage site recognized by the GFLV proteinase between the 28K and the 38K proteins in the 66K protein in addition to the Arg605/Gly606 site between the 38K protein and the coat protein.

Biologically active transcripts from cloned cDNA of genomic grapevine fanleaf nepovirus RNAs.

Transcripts were produced in vitro by run-off transcription from full-length cDNA of RNA1 and RNA2 of grapevine fanleaf nepovirus (GFLV; isolate F13) cloned downstream from a bacteriophage RNA polymerase promoter. These transcripts, which possess a 5' terminal cap structure and a non-viral G residue instead of the naturally occurring genome-linked viral protein (VPg), are infectious to Chenopodium quinoa protoplasts when inoculated by electroporation. Synthetic RNA1 alone replicated in protoplasts. Inoculation of C. quinoa plants with synthetic RNA1 plus RNA2 produced symptoms similar to, but weaker, than those observed in plants infected with natural GFLV 6 to 8 days post-inoculation. Co-inoculated RNA1 and RNA2 were able to replicate and spread systemically in plants but RNA1 alone produced no symptoms and was not detected in non-inoculated leaves, suggesting that virus spread requires RNA2. Analysis of the genomic RNAs in plants infected with transcripts showed that the non-viral G at their 5' ends was not retained in the progeny.

Location of the replication determinants of the satellite RNA associated with grapevine fanleaf nepovirus (strain F13).

A large satellite RNA of 1114 nucleotides, named RNA3, is always found associated with the genomic RNAs of grapevine fanleaf virus, isolate F13 (GFLV-F13). RNA3 encodes a non-structural protein (P3) of M(r) 37K to which no function has previously been assigned. Full-length cDNA clones of RNA3 were mutated in the 5' and 3' non-coding regions and in the 37K open reading frame. The ability of transcripts obtained from these clones to be replicated was investigated by protoplast infection in the presence of a helper virus. We demonstrate that the 5' and 3' non-coding regions as well as the satellite-encoded P3 protein are essential for replication of the GFLV-F13 satellite RNA. Our results suggest that two hydrophobic regions located at the N- and C-extremity of P3 and a zinc-finger motif near the C-terminal extremity of P3 are probably involved in the replication of this satellite. Analysis of the in vitro translation products from transcripts of RNA3 clones of different lengths indicates that the double band formed by P3 could result from phosphorylation of a part of this protein.

Cloning and in vitro characterization of the grapevine fanleaf virus proteinase cistron.

The region of the genomic RNA-1 from grapevine fanleaf virus isolate F13 (GFLV-F13), containing the proteinase cistron and flanking sequences (nucleotides 3894 to 4789) of the GFLV polyprotein, was modified by PCR mutagenesis to create a start codon and cloned in a transcription vector. The transcripts from the resulting clone (pVP7) produced, upon translation in rabbit reticulocyte lysate, a 37.8-kDa protein which was subsequently cleaved to a stable 28-kDa product. Autocleavage was maximal at pH 7.0-8.5 and at 30 degrees. Inhibition of the activity was greater than 80% when translation was performed in the wheat germ system. In rabbit reticulocyte lysate, inhibition was also obtained with PMSF, EDTA, E-64, Ca+2, Zn+2, and Co+2. The pVP7 translation product acts in cis, in the case of its autocleavage, or in trans in the processing of the viral 122-kDa polyprotein from GFLV RNA-2 into a 66-kDa protein and the 56-kDa coat protein. The carboxy extremity of the complete pVP7 translation product, encoded by nucleotides 4633 to 4789 of RNA-1, was not required for the proteinase activity, at least in trans.

Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1.

The nucleotide sequence of the genomic RNA1, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-F13) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an Mr of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picronavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-F13 RNA1. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the substrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of Mr 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease cofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.

Primary structure and location of the genome-linked protein (VPg) of grapevine fanleaf nepovirus.

The genome linked protein VPg covalently linked to the RNAs of grapevine fanleaf nepovirus has been sequenced. The VPg (Mr = 2931) composed of 24 residues is linked by its N-terminal Ser beta-OH group to the viral RNAs. The VPg mapped from residues 1218 to 1241 of the 253K polyprotein encoded by GFLV RNA1.

In vitro expression of a chimeric coat protein gene from Grapevine Fanleaf virus (strain F 13).

The coat protein (CP) cistron of Grapevine Fanleaf virus strain F13 (GFLV-F13) has been located in the C-terminal region of the 122k polyprotein encoded by the genomic RNA 2 [Serghini et al. (1990) J. Gen. Virol. 71: 1433-1441]. A chimeric CP gene of GFLV-F13 including a short sequence corresponding to 3 restriction sites, the leader sequence of the GFLV-F13 satellite RNA and an initiation codon was constructed. Transcripts from this construct were translated in wheat germ extract with equal efficiency to form a 56k protein which comigrates on PAGE with the GFLV-F13 CP and a protein of 52k. Both species react with GFLV-F13 CP-specific antibodies. Deletions in the 5' region of the CP gene show that the 56k protein is initiated at the first AUG after the satellite leader and the 52k protein at the second in-frame AUG. Transcripts with a 142 nt deletion including the two AUG codons from the 5' end of the CP gene are not efficiently expressed in vitro, no major translation product being detected.

RNA2 of grapevine fanleaf virus: sequence analysis and coat protein cistron location.

The nucleotide sequence of the genomic RNA2 (3774 nucleotides) of grapevine fanleaf virus strain F13 was determined from overlapping cDNA clones and its genetic organization was deduced. Two rapid and efficient methods were used for cDNA cloning of the 5' region of RNA2. The complete sequence contained only one long open reading frame of 3555 nucleotides (1184 codons, 131K product). The analysis of the N-terminal sequence of purified coat protein (CP) and identification of its C-terminal residue have allowed the CP cistron to be precisely positioned within the polyprotein. The CP produced by proteolytic cleavage at the Arg/Gly site between residues 680 and 681 contains 504 amino acids (Mr 56019) and has hydrophobic properties. The Arg/Gly cleavage site deduced by N-terminal amino acid sequence analysis is the first for a nepovirus coat protein and for plant viruses expressing their genomic RNAs by polyprotein synthesis. Comparison of GFLV RNA2 with M RNA of cowpea mosaic comovirus and with RNA2 of two closely related nepoviruses, tomato black ring virus and Hungarian grapevine chrome mosaic virus, showed strong similarities among the 3' non-coding regions but less similarity among the 5' end non-coding sequences than reported among other nepovirus RNAs.

The nucleotide sequence of satellite RNA in grapevine fanleaf virus, strain F13.

The nucleotide sequence of cDNA copies of grapevine fanleaf virus (strain F13) satellite RNA has been determined. The primary structure obtained was 1114 nucleotides in length, excluding the poly(A) tail, and contained only one long open reading frame encoding a 341 residue, highly hydrophilic polypeptide of Mr37275. The coding sequence was bordered by a leader of 14 nucleotides and a 3'-terminal non-coding region of 74 nucleotides. No homology has been found with small satellite RNAs associated with other nepoviruses. Two limited homologies of eight nucleotides have been detected between the satellite RNA in grapevine fanleaf virus and those in tomato black ring virus, and a consensus sequence U.G/UGAAAAU/AU/AU/A at the 5' end of nepovirus RNAs is reported. A less extended consensus exists in this region in comovirus and picornavirus RNA.

Competitive multiplication of RNA3 species of different strains of alfalfa mosaic virus.

Competition between RNA3 from alfalfa mosaic virus (AlMV) strain S (RNA3-S), strain B (RNA3-B) and strain 425L (RNA3-L) was studied. The identification of the RNA3 species multiplying in infected leaves was possible since the RNA3 5' non-coding leader sequences in strains S, B and 425L differ in length. RNA3 present in total RNA from infected tobacco leaves was detected, and strains were identified from the length of the cDNA reverse-transcribed from RNA primed with a specific oligonucleotide. In competition experiments the inoculum, containing known amounts of RNA1, 2, 3 and 4 of one strain, was complemented with various amounts of heterologous RNA3 and inoculated to a systemic host. It is shown that RNA3-S was better replicated in vivo by the AlMV replicase of strain B than was RNA3-B itself, and to a lesser extent better replicated by the AlMV replicase of strain L than was RNA-L. Comparison of genetic information carried by the RNA3 species present in the inoculum suggests that the more efficient multiplication of RNA3-S is related to the structure of the leader sequence of RNA3-S.

A simple gene-expression system for the small subunit of ribulose bisphosphate carboxylase in leaves ofNicotiana sylvestris.

InNicotiana sylvestris only four transcripts coding for the small subunit of RUBISCO are present in leaves. They are very closely related as they are identical in the nucleotide sequence of the non-coding regions and show only three silent point differences in the region coding for the mature peptide.The main difference among these four transcripts lies in the length of the non-coding regions. Half of the SmRNA population as confirmed by direct RNA sequencing has an additional nucleotide sequence in the leader region. Two cDNAs have an additional nucleotide sequence at the end of the 3' non-coding region. Based on these criteria the transcripts were classified into two groups:.group I has a 73-nucleotide-long leader sequence and the nucleotides T, A and C at position 327, 432 and 519 in the coding region..group II has a 60-nucleotide-long leader sequence and the nucleotides C, G and T at these positions in the coding region.The two cDNAs showing a difference in the length of the 3' non-coding region belong to group II.The study of all these transcripts argues for the possibility that only two families of genes are expressed in leaves ofN. sylvestris.

Complete sequence of one of the mRNAs coding for the small subunit of ribulose bisphosphate carboxylase of Nicotiana sylvestris.

The combination of cDNA and RNA sequencing techniques has enabled determination of the complete sequence of one of the mRNAs coding for the precursor of the small subunit of ribulose bisphosphate carboxylase of Nicotiana sylvestris. In this 898-nucleotide-long mRNA, 540 nucleotides code for the entire 180-amino-acid-long precursor polypeptide consisting of the 57-amino acid-long transit peptide and the 123-amino-acid-long mature protein, while 60 and 195 nucleotides belong to the 5' and 3' noncoding flanking regions, respectively. The 5' end, which is very rich in AG residues, contains several direct and indirect repeated sequences, and a possible hairpin structure. The 3' end, terminated by a 103-nucleotide-long poly-A tail, is very rich in AU residues but does not contain the classical polyadenylation signal sequence.

Complete nucleotide sequence of RNA 3 from alfalfa mosaic virus, strain S.

We report the sequence of RNA 3 from strain S of Alfalfa mosaic virus (2,055 nucleotides). This RNA codes for a 32.4 kd protein (P3) and for the 24 kd coat protein (P4). The largest part of the sequence was established using RNA sequencing methods. The completion of the sequence in the region coding for P3 was achieved with cloned cDNA synthesized after priming at internal sites of RNA 3. Comparison of the RNA sequences coding P3 and P4 proteins in strain S with those reported in the literature for strain 425 revealed a higher amino acid substitution rate (3%) for P3 than for P4 (congruent to 1%) despite a similar average base substitution of 3-4% in these regions. In P3, two out of nine amino acid changes occur in hydrophilic regions. The amino acid changes in P4 do not modify the local hydrophilicity distribution. The intercistronic region displays a low degree of base substitution (2%) when compared with the untranslated 3'-end region (3.6%) or the 5'-end leader region (8%), the average substitution rate being 3.2%.

Expression of the gene coding for the small subunit of ribulosebisphosphate carboxylase during differentiation of tobacco plant protoplasts.

A hybridization probe was used to study the regulation of expression of the gene coding for the small subunit of ribulose 1,5-bisphosphate carboxylase, during functional differentiation of protoplasts. A library of cDNA from poly(A)-containing RNA extracted from specially treated tobacco leaves was constructed in the plasmid pBR322 by blunt-end ligation. This library was screened by colony hybridization with 32P-labelled cDNA prepared from mRNA coding for the precursor of the small subunit. A positive colony was identified containing recombinant plasmids with a nucleotide sequence homologous to this mRNA. These plasmids, bound to diazobenzyloxymethylated cellulose paper, were then used as a hybridization probe. The results showed unambiguously that the small subunit was not transcribed in protoplasts but was transcribed in undifferentiated white and chlorophyll-containing green callus cultures derived from protoplasts. The discrepancy between these results and those obtained with classical techniques is discussed.

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.

Aminoacylation and messenger functions of eggplant mosaic virus RNA.

The predominant RNA of eggplant mosaic virus (EMV) was found to have a molecular weight of 1.9 x 10(6) by formamide gel electrophoresis. Electrophoretic analysis of virion protein under dissociating conditions revealed two polypeptides, a major component of 21,000 daltons, and a minor component of 22,000 daltons. Both peptides were present in translation products coded by RNA isolated from virions, but the proportion of the 22,000-dalton peptide was higher in products synthesized using RNA isolated from EMV-infected Datura leaves as messenger. Since the viral RNA showed a marked tendency to aggregate, it is possible that these polypeptides were translated from trace amounts of small EMV RNAs present as contaminants of the 1.9 x 10(5)-dalton genomic RNA. Although the addition of tRNA from infected or healthy Datura leaves, or from wheat germ, stimulated amino acid incorporation, no changes were discerned in the profile of cell-free translation products after electrophoretic separation. Nonaminoacylated and valylated EMV RNA stimulated similar levels of amino acid incorporation, and the translation products appeared identical. Valine bound to genomic EMV RNA was not donated during protein synthesis.